Project Summary Hospital-acquired infections are a major threat to public health, impacting 2 million patients and causing at least 90,000 deaths annually. Sepsis is a common complication in patients with hospital-acquired infections and the leading cause of death in non-cardiac intensive care units (ICU). Attempts at treating hospital-acquired infections and sepsis have proven exceedingly difficult and patients that survive sepsis suffer long-term physical and cognitive disabilities and a high 1-year mortality rate. Therefore, new strategies are needed to decrease the burden of hospital-acquired infections and sepsis. Immunotherapy aimed at inducing innate immune memory provides a way of achieving that goal. Recent studies show that innate immune cells can retain memory of prior pathogen exposure and are primed to elicit a robust, broad-spectrum antimicrobial response to subsequent infection. Treatment with TLR4 ligands, such as monophosphoryl lipid A (MPLA), confers innate immune memory and resistance to a broad array of clinically important pathogens that persists for more than 2 weeks. We propose that the appropriate application of TLR4 ligand-based immunotherapy to induce innate immune memory has significant potential to reduce the burden of hospital-acquired infections and sepsis. Macrophages are the foundation for development of innate immune memory. Recent evidence indicates that remodeling of macrophage metabolism is central to the induction of innate immune memory. Priming with TLR4 ligands induces a macrophage metabolic phenotype characterized by increased glycolysis, oxidative metabolism and mitochondrial biogenesis with increased citric acid cycle flux and associated increases in immunoresponsive gene 1 (Irg1) expression and itaconate production. We hypothesize that macrophage metabolic remodeling and the increased production of Irg1 and itaconate are essential to generating innate immune memory. To define the underlying biology, we will: (1). Determine how Irg1 and itaconate drive TLR4 agonist-induced innate immune memory in macrophages; (2). Define the importance of Irg1, itaconate and Nrf2 as regulators of the host response to infection with common hospital-acquired pathogens after TLR4 agonist treatment in vivo.; (3). Define the intracellular signaling pathways driving mitochondrial biogenesis, Irg1 expression and itaconate production in TLR4 agonist-primed macrophages; (4). Determine the ability of diverse microbial ligands to induce macrophage mitochondrial biogenesis, reprogram mitochondrial metabolism and function and induce innate immune memory. We will test the hypothesis that, like TLR4 agonists, microbial ligands such as peptidoglycan, CpG ODN and ?-glucan, have the ability to reprogram macrophage metabolism and induce a memory phenotype characterized by mitochondrial biogenesis, increased citric acid cycle flux, increased Irg1 expression and itaconate production with associated enhancement of antimicrobial functions.